Background/Aims: The role of the β3-adrenergic receptor (β3-AR) agonist BRL37344 in atrial fibrillation (AF) structural remodeling and the underlying mechanisms as a therapeutic target were investigated. Methods: Four groups of dogs were evaluated: sham, pacing, β3-AR agonist BRL37344 (β3-AGO), and β3-AR antagonist L748337 (β3-ANT) groups. Dogs in the pacing, β3-AGO and β3-ANT groups were subjected to rapid atrial pacing for four weeks. Atrial structure and function, AF inducibility and duration, atrial myocyte apoptosis and interstitial fibrosis were assessed. Atrial superoxide anions were evaluated by fluorescence microscopy and colorimetric assays. Cardiac nitrate+nitrite levels were used to assess nitric oxide (NO) production. Protein and mRNA expression of β3-AR, neuronal NO synthase (nNOS), inducible NO synthase (iNOS), endothelial NO synthase (eNOS) and guanosine triphosphate cyclohydrolase-1 (GCH-1) as well as tetrahydrobiopterin (BH4) levels were measured. Results: β3-AR was up-regulated in AF. Stimulation of β3-AR significantly increased atrial myocyte apoptosis, fibrosis and atrial dilatation, resulting in increased AF induction and prolonged duration. These effects were attenuated by β3-ANT. Moreover, β3-AGO reduced BH4 and NO production and increased superoxide production, which was inhibited by the specific iNOS inhibitor, 1400w β3-AGO also increased iNOS but decreased eNOS and had no effect on nNOS expression in AF. Conclusions: β3-AR stimulation resulted in atrial structural remodeling by increasing iNOS uncoupling and related oxidative stress. β3-AR up-regulation and iNOS uncoupling might be underlying AF therapeutic targets.

Atrial fibrillation (AF) is the most common self-sustained cardiac arrhythmia closely associated with stroke and heart failure, and its prevalence is rapidly increasing in the aging population [1]. AF is self-perpetuating because AF stimulates atrial electrical, structural and metabolic remodeling [2,3,4]. Atrial remodeling, in turn contributes to AF development and maintenance, creating a vicious cycle [5,6]. After AF cardioversion to sinus rhythm, atrial electrical changes can be completely recovered within a few days, but structural damages may not be fully reversed. Moreover, structural remodeling breaks myocardial continuity, slows electrical conduction, and damages myocardial energy metabolism [4,7]. Therefore, atrial structural remodeling is vital in AF development as an important substrate. However, the pathophysiology underlying atrial structural remodeling remains poorly understood.

β-adrenoceptors (β-ARs), including β1, β2 and β3 subtypes, are important modulators in cardiovascular function, β1- and β2- ARs classically mediate the effects of catecholamines on cardiac contraction and vascular smooth muscle relaxation [8]. However, β3-AR differs from the β1- and β2 subtypes in its molecular structure and pharmacological profile [9]. Although many studies have demonstrated that β3-AR upregulation contributes to dysfunction in heart failure [10,11], few reports have described the potential role of β3-AR in AF. A recent study revealed that β3-AR activation induced atrial electrical remodeling in rapid paced atrial myocytes [12], suggesting that β3-AR promotes AF development. However, the role of β3-AR in atrial structural remodeling associated with AF remains unclear and might present a new therapeutic target.

Oxidative stress is paramount when promoting AF pathogenesis by affecting atrial structural remodeling [13]. Reactive oxygen species (ROS) generated from nitric oxide synthase (NOS) uncoupling may be an underlying contributor [14]. NO is synthesized from L-arginine and oxygen by NOS using tetrahydrobiopterin (BH4) as a cofactor. Under conditions of low BH4 bioavailability relative to NOS, NOS is uncoupled to produce superoxide anion (O2-) instead of NO (Fig. 1) [14]. There are three isoforms of NOS: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) [15]. It is well established that negative inotropy associated with β3-AR results from the eNOS-NO pathway. However, it remains unclear whether the effect of β3-AR on atrial structural remodeling in AF is related to NOS uncoupling.

Fig. 1

Left: In the process of nitric oxide (NO) production, nitric oxide synthase (NOS) enzymes require the substrates L-arginine and molecular oxygen (O2) and the cofactor tetrahydro-biopterin (BH4). Right: Reduced BH4 bioavailability relative to NOS protein results in “uncoupled” NOS characterized by the production of superoxide (O2-).

Fig. 1

Left: In the process of nitric oxide (NO) production, nitric oxide synthase (NOS) enzymes require the substrates L-arginine and molecular oxygen (O2) and the cofactor tetrahydro-biopterin (BH4). Right: Reduced BH4 bioavailability relative to NOS protein results in “uncoupled” NOS characterized by the production of superoxide (O2-).

Close modal

Here, we investigated the potential role of β3-AR in AF canine models induced by rapid atrial pacing and tested the hypothesis that the preferential β3-AR agonist BRL37344 exacerbates atrial structural remodeling in AF by increasing oxidative stress from iNOS uncoupling.

Animal Welfare and Ethical Statement

All animal procedures were approved by the Experimental Animal Ethics Committee at the Harbin Medical University. All procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996).

Animals

A total of 24 male and female mongrel dogs (2 - 3 years ofage, 15 - 25 kg) were randomized into four groups: sham group (sham surgery) (n = 6); pacing group (no drug) (n = 6); β3-AGO group (β3-AR agonist, BRL37344) (n = 6); and β3-ANT group (β3-AR antagonist, L748337) (n = 6). After adequate anesthesia with sodium pentobarbital (25 mg/kg i.v.), the dogs were rapidly intubated and mechanically ventilated. Thoracotomy was performed from the right fourth intercostal space. A thin silicon lead was implanted into the right atrial free wall and affixed to a pacemaker (Harbin Science and Technology University, China). One week post-surgery, the dogs in the pacing, β3-AGO and β3-ANT groups were paced at 600 beats per minute (bpm) for four weeks according to a previous study [16]. In the β3-AGO and β3-ANT groups, the dogs were continuously given an intraperitoneal injection of BRL37344 (1.5 µg/kg/h, Sigma-Aldrich B169) and L748337 (2 µg/kg/h, Sigma-Aldrich L7045), respectively, by an osmotic pump for four weeks during the rapid atrial pacing phase. All experiments were performed in the presence of nadolol, an antagonist for β1- and β2-ARs (1 mg/kg, i.p. injection; cat. no. N1892; Sigma-Aldrich). A surface electrocardiogram was recorded daily to ensure sustained atrial pacing at 600 bpm.

Echocardiography

Left atrium (LA) and left atrial appendage (LAA) structure and function were serially assessed by transthoracic and transesophageal echocardiography (Philips 7500, Washington, USA) prior to surgery and heart removal. Parameters related to the LA maximal volume (LAVmax), LAA maximal volume (LAAVmax), LA minimal volume (LAVmin), and LAA minimal volume (LAAVmin) were recorded. The LA ejection fraction was determined as (LAEF) = (LAVmax - LAVmin) ⁄ LAVmax x 100%, and the LAA ejection fraction was determined as (LAAEF) = (LAAVmax -LAAVmin)/LAAVmaxx 100%.

Electrophysiological assessment of AF

AF inducibility and duration were measured at baseline and at the end of the four-week protocol. AF inducibility and duration were assessed using 10 Hz stimuli (burst pacing at 10 Hz for 10 s) at five-minute intervals with a pacing cycle length of 100 ms. AF was defined as a rapid and irregular atrial rhythm. AF lasting more than 30 minutes was terminated by direct current electrical cardioversion, and the AF duration was recorded for 30 minutes.

Histological analysis of cellular morphometry

Dogs were anesthetized, and hearts were rapidly excised. Current evidence suggests that the left atrium dominates the occurrence of AF [17,18]. Therefore, left atrial samples adjacent to the left atrium appendage were collected, snap-frozen in liquid nitrogen and then divided into four portions: frozen sections for determination of superoxide (O2· ) immediately after excision; tissue fixed with 10% paraformaldehyde and embedded in paraffin for morphological evaluation; tissue fixed in glutaraldehyde at 4°C for electron microscopy; and samples frozen in liquid nitrogen for subsequent assays. Sections (5 µm) of the atrium were stained using a Masson trichrome and imaged using light microscopy. The interstitial collagen volume fraction (CVF) was used to evaluate fibrosis. After staining, collagen fibers and cardiomyocytes were stained blue and red, respectively. One section was selected from each sample, and five randomly selected fields from each section were evaluated. The mean area percentage of the collagen fibers was recorded as the collagen volume fraction (CVF%). Perivascular collagen was not included in the quantification. Sections were evaluated by two professionals who were blinded to the treatment. Digital images were analyzed using Image-Pro Plus 6.0 software.

Ultrathin sections (50-100 nm) pretreated with gluteraldehyde were prepared for electron microscopy (HITACHI, H7650, Japan) by two observers blinded to the treatments. Sections were prepared according to the method of Zhao etal. [19],

Terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) analysis

Tissues embedded in paraffin were sectioned to 7-µm thickness, dewaxed and rehydrated prior to TUNEL staining. TUNEL staining was performed using an In Situ Cell Death Detection kit (Roche Applied Science) according to the manufacturer's instructions. Tissue sections were incubated in 0.1 M citrate buffer and 0.1% Triton, pH 6.0, and subsequently exposed to 350 W of microwave irradiation for 5 minutes. The sections were then rapidly cooled using distilled water (20 - 25°C) and rinsed with phosphate buffered saline (PBS) at 15 - 25°C. Subsequently, 50 µl of TUNEL reaction mixture was added to the sections and incubated for 60 minutes at 37°C. Finally, slides were rinsed three times with PBS. Negative control sections were incubated with 50 µl of labeling solution. Positive control sections were incubated with DNAse I (Sigma-Aldrich). Sections were subsequently imaged and microscopically analyzed for TUNEL-positive cells. The percentage of TUNEL-positive nuclei was calculated and recorded as the apoptotic index (AI%). Five randomly selected fields per section corresponding to approximately 300 cells were examined per treatment condition at high magnification (200 x).

Western blot (WB) analysis of β-3AR, nNOS, iNOS, eNOS and guanosine triphosphate cyclohydrolase-1 (GCH-1) protein expression

Protein samples were extracted from tissues, and a BCA protein assay kit was used to calculate protein concentrations. A total of 50 µg of protein for β-3AR, iNOS, eNOS and GCH-1 and 150 µg protein for nNOS was loaded and separated on SDS gels (8-12%) followed by blotting onto polyvinylidene difluoride (PVDF) membranes. The membranes were subsequently blocked with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) (0.05% Tween 20, pH 7.4). Membranes were incubated overnight at 4°C with primary antibodies in TBST and subsequently washed in TBST; the membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at 37°C. The primary antibodies included rabbit anti-β3-AR (1:500, Abcam, ab76249), nNOS (1:100, Santa Cruz, sc648), iNOS (1:150, Abcam, ab3523), synthetic peptide corresponding to aa598-612 (P598YNSSPRPEQHKSYK612C) of bovine eNOS (1:1000, Biomol, ALX-210-505) and mouse anti-GCH-1 (l:400, Santa Cruz, scl34574). Glyceraldehyde phosphate dehydrogenase (GAPDH) levels were measured as an internal control with anti-GAPDH (1:1000) antibodies (Abcam, Cambridge, MA, USA). Immunoreactive bands were detected, and the signal intensities were quantified using a chemiluminescent protein detection system (Bio-Rad Laboratories).

Real time polymerase chain reaction (RT-PCR) analysis to detect β3-AR, nNOS, iNOS, eNOS and GCH-1 mRNA expression

Total RNA was isolated using Trizol according to the manufacturer's instructions. Reverse transcription was performed in a 20 µl reaction mixture with SYBR®Green qPCR reagents, TaKaRa PrimeScript TM RT reagent Kit and gDNA Eraser (Code No. RR047A) according to the manufacturer's recommendations. Quantitative RT-PCR was performed using an ABI 7500 Real Time PCR system (Applied Biosystems). Canine β3-AR, nNOS, iNOS, eNOS, and GCH-1 steady state mRNA levels were measured. Specific primers used to amplify these genes are listed in Table 1. GAPDH was used as an internal control.

Table 1

Primers for real-time PCR

Primers for real-time PCR
Primers for real-time PCR

Fluorescence measurement ofsuperoxide anion (O2·-)

The cell-permeable dihydroethidium (DHE) is oxidized to fluorescent hydroethidium (HE) by O2·-, which is then intercalated into DNA. The generation of O2·- in atrial tissue was determined based on DHE fluorescence, as previously described [20] and according to the manufacturer's instructions (GENMED SCIENTIFICS INC. USA, 10111.2 v. A).

Fresh left atrial samples collected within 1 hour after surgery were placed in ice-cold saline and then embedded for cyrosectioning at the optimal cutting temperature (-20°C). Each sample was cut into five 10-µm-thick sections and then placed on glass slides. Sections were washed with 0.5 ml of Reagent A followed by topical application of a DHE stain working solution (10 µM, 0.2 ml). Sections were then incubated for 20 minutes at 37°C in a light-protected chamber and subsequently washed with 0.5 ml of reagent A to remove any unbound DHE. Sections were then fixed, secured with a coverslip, and imaged using a laser scanning confocal microscope (ZEISS, LSM510.META). Fluorescence intensity was quantified using Image-Pro Plus 6.0 software.

Colorimetric assay of superoxide anions (O2·-)

To further measure O2·- levels and evaluate the effect of the iNOS inhibitor, 1400w, a colorimetric assay was used to measure O2·- in atrial tissues. The assay was performed on fresh left atrial tissues, which were collected within 1 hour after surgery. Canine atrial tissue (50 mg) that was directly adjacent to the LA appendage was used for the assay. Tissues were subsequently homogenized, and proteins were extracted. The extracts were divided into two aliquots (50 µl each), and the concentrations were measured. Nicotinamide adenine dinucleotide phosphate (NADPH) solution (1X) was added to tissue homogenates (one of the 50 µl aliquots) after 30 minutes at 4ºC to a total volume of 1 ml, which resulted in a final NAPDH concentration of 100 µM. The other tissue homogenate (the other 50 µl aliquot) was pre-incubated with 100 µM iNOS inhibitor, 1400w in a total volume of 1 ml for 30 minutes at 4ºC. Staining was performed according to the GENMED Colorimetric assay kit instructions (GENMED SCIENTIFICS INC. U.S.A, 10096.2v.A). The optical density (OD) was determined at a wavelength of 580 nm using a microplate reader. The results were subtracted from the total OD to determine iNOS-dependent O2-· generation.

Measurement of cardiac NO production

Cardiac NO levels were determined using a two-step measurement of total nitrate+nitrite concentrations and the Nitrate/Nitrite Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, MI, Item No.780001). Atrial tissues (60 mg) were homogenized in PBS at pH 7.4 and subsequently centrifuged at 10,000 x g for 20 minutes. Supernatants were diluted to 6 ml and ultracentrifuged at 100,000 x g for 30 minutes (BECKMAN COULTER, Optima™L-100XP Ultracentrifuge). Tissue homogenates (80 µl) were generated and used in triplicate. The absorbance of samples was between 0.05 - 1.2 absorbance units. A total of 80 µl of the tissue homogenate was added to the sample wells, and 200 µl of assay buffer was added to blank wells as a negative control. Subsequently, the enzyme cofactor mixture (10 µl) and nitrate reductase mixture (10 µl) were added to each well (standards and experimental samples). The plate was then incubated at room temperature for 1 hour. Subsequently, Griess Reagent R1 (50 µl) and Griess Reagent R2 (50 µl) were added to each of the wells. The color was then allowed to develop for 10 minutes at room temperature. The absorbance was read at 540 nm using a Bio-Rad iMark™ microplate reader.

High-performance liquid chromatography (HPLC) for the assessment of BH4

Atrial tissue (100 mg) was analyzed using HPLC. Tissue was homogenized and diluted to 1000 µl prior to processing (500 µl each). Protein concentrations were assessed using the BCA method. Samples (450 µl) were incubated with a 50 µl acid solution (1.5 M HClO4 and 2 M H3PO4 mixture of equal proportions) at 4ºC and subsequently centrifuged at 13,000 g/min for 10 minutes. The supernatant (450 µl) was transferred to a new Eppendorftube and incubated with 50 µl of acidic iodine solution (1% iodine + 2% KI dissolved inl M HCl) at room temperature in the dark for 30 minutes. Subsequently, 25 µl of freshly prepared ascorbic acid (20 mg/ml) was added at 4ºC and centrifuged at 13,000 g/min for 10 minutes. A portion of the supernatant (40 µl) was used to determine the total acid oxidation levels of biopterins (BH4 + BH2 + biopterin). Simultaneously, 400 µl of the same protein extract was incubated with a mixture of 50 µl of 1 M NaOH and 50 µl of alkaline iodine (1% iodine + 2% KI dissolved in 1 M NaOH) in the dark at room temperature for 60 minutes. Subsequently, 100 µl of 1 M H3PO4 and 25 µl of a freshly prepared ascorbic acid solution (20 mg/ml) were added to the protein extracts at 4°C and centrifuged at 13,000 g/min for 10 minutes. A portion of the supernatant (40 µl) was then used to measure biopterins that excluded BH4 (BH2 + biopterin). After oxidation (either in acidic or alkaline solution), the sample was immediately used for HPLC analysis. The mobile phase of the HPLC consisted of methanol/water (10/90) at a flow rate of 10 ml/min. Results were obtained by comparison to a standard curve and the results from analytical standards, which were obtained from Sigma-Aldrich (B-1527 and 37272) and diluted to various concentrations (1, 10, 20, 50 and 100 nM). BH4 levels were calculated as the difference between the two measurements (acid and alkaline oxidation) as described by Takeshi and colleagues [21]. Detection of BH4 was assessed by fluorescence detection at excitation and emission wavelengths of 350 nm and 440 nm, respectively, using a Waters 515 HPLC pump, 717 plus autosamplers, and a 2475 multi λ fluorescence instrument.

Statistics

All data are expressed as the mean ± standard deviation. Echocardiographic, electrophysiological data and iNOS-dependent O2-· generation were compared using repeated measures analysis of variance (RM-ANOVA). Group data were compared using a one-way ANOVA with a Tukey's post-hoc test for multiple comparisons. A P value of less than 0.05 was considered statistically significant. GraphPad Prism 5.0 and 6.0 (La Jolla, CA) were used for the statistical analysis.

Upregulatìon of β3-AR in canine AF models was induced by rapid atrial pacing

RT-PCR analysis and WB results revealed that β3-AR mRNA and protein levels were significantly higher in the pacing group compared to the sham group (P < 0.01). Furthermore, the expression of β3-AR mRNA and protein was significantly increased in the β3-AR agonist (BRL37344) β3-AGO group (P < 0.01) and decreased in the β3-AR antagonist (L748337) β3-ANT group (P < 0.05) compared to the pacing control group (Fig. 2A, B, C).

Fig. 2

β3-AR mRNA and protein levels in the left atrium. (A) β3-AR mRNA expression in the sham, pacing, β3-AGO and β3-ANT groups. (B) Western bolt analysis of β3-AR protein levels using specific β3-AR antibodies. GAPDH was used as a loading control. (C) Quantitative analysis of β3-AR protein levels relative to GAPDH levels. **P < 0.01 vs. sham group, ##P < 0.01, #P < 0.05 vs. pacing group. β3-AGO refers to the β3-AR agonist BRL37344 group, and β3-ANT refers to the β3-AR antagonist L748337 group.

Fig. 2

β3-AR mRNA and protein levels in the left atrium. (A) β3-AR mRNA expression in the sham, pacing, β3-AGO and β3-ANT groups. (B) Western bolt analysis of β3-AR protein levels using specific β3-AR antibodies. GAPDH was used as a loading control. (C) Quantitative analysis of β3-AR protein levels relative to GAPDH levels. **P < 0.01 vs. sham group, ##P < 0.01, #P < 0.05 vs. pacing group. β3-AGO refers to the β3-AR agonist BRL37344 group, and β3-ANT refers to the β3-AR antagonist L748337 group.

Close modal

β3-AR stimulation induced and maintained AF in canines

In the pacing groups, AF inducibility (P < 0.01) and duration (P < 0.01) were markedly increased compared to the sham group. Moreover, the β3-AR agonist (BRL37344; β3-AGO) further increased AF inducibility (P < 0.01) and prolonged AF duration (P < 0.01) compared to the pacing alone group. Interestingly, the β3-AR antagonist (L748337) significantly inhibited (P < 0.01) AF induction and maintenance (Table 2).

Table 2

Electrophysiological evaluation of canine atria before and after rapid atrial pacing. AF cases refer to the number of dogs successfully induced (10 inductions per dog) to develop AF during electrophysiological examination in each group. AF frequency refers to the total number of AF episodes that were induced during electrophysiological examination in all dogs within a group (with the understanding that AF may have been repeatedly induced in the same dog within the scope of its 10 inductions). AF inducibility is calculated as AF frequency/total stimulations, eg. 60. *P < 0.05, **P < 0.01 vs. baseline; #P < 0.05, ##P < 0.01 vs. sham group; †P < 0.05, ††P < 0.01 vs. pacing group

Electrophysiological evaluation of canine atria before and after rapid atrial pacing. AF cases refer to the number of dogs successfully induced (10 inductions per dog) to develop AF during electrophysiological examination in each group. AF frequency refers to the total number of AF episodes that were induced during electrophysiological examination in all dogs within a group (with the understanding that AF may have been repeatedly induced in the same dog within the scope of its 10 inductions). AF inducibility is calculated as AF frequency/total stimulations, eg. 60. *P < 0.05, **P < 0.01 vs. baseline; #P < 0.05, ##P < 0.01 vs. sham group; †P < 0.05, ††P < 0.01 vs. pacing group
Electrophysiological evaluation of canine atria before and after rapid atrial pacing. AF cases refer to the number of dogs successfully induced (10 inductions per dog) to develop AF during electrophysiological examination in each group. AF frequency refers to the total number of AF episodes that were induced during electrophysiological examination in all dogs within a group (with the understanding that AF may have been repeatedly induced in the same dog within the scope of its 10 inductions). AF inducibility is calculated as AF frequency/total stimulations, eg. 60. *P < 0.05, **P < 0.01 vs. baseline; #P < 0.05, ##P < 0.01 vs. sham group; †P < 0.05, ††P < 0.01 vs. pacing group

β3-AR stimulation resulted in atrial dilation and exacerbated atrial dysfunction in the rapid pacing canine model of AF

Compared to the sham groups, LAVmax, LAVmin, LAAVmax, and LAAVmin were significantly increased in the pacing group (P < 0.01), while LAEF and LAAEF were decreased (P < 0.01). Compared to the pacing group, the LAVmax, LAVmin, LAAVmax and LAAVmin were significantly increased in the β3-AGO group (P < 0.01), while LAEF and LAAEF were decreased (P < 0.01). Interestingly, the β3-AR antagonist L748337 abrogated the effects of atrial dilation and dysfunction in the canine pacing model (P < 0.05) (Table 3).

Table 3

Atrial dimensions and function in canines before and after a four-week rapid pacing protocol. *P < 0.05 ‚ **P < 0.01 vs. baseline, ##P < 0.01 vs. sham group, †P < 0.05 ‚ ‡P < 0.01 vs. pacing group. LAV max, LA maximal volume; LAVmin, LA minimal volume; LAEF, LA ejection fraction; LAAVmax, LAA maximal volume; LAAVmin, LAA minimal volume; LAAEF, LAA ejection fraction. 5 weeks refers to 5 weeks post-operation

Atrial dimensions and function in canines before and after a four-week rapid pacing protocol. *P < 0.05 ‚ **P < 0.01 vs. baseline, ##P < 0.01 vs. sham group, †P < 0.05 ‚ ‡P < 0.01 vs. pacing group. LAV max, LA maximal volume; LAVmin, LA minimal volume; LAEF, LA ejection fraction; LAAVmax, LAA maximal volume; LAAVmin, LAA minimal volume; LAAEF, LAA ejection fraction. 5 weeks refers to 5 weeks post-operation
Atrial dimensions and function in canines before and after a four-week rapid pacing protocol. *P < 0.05 ‚ **P < 0.01 vs. baseline, ##P < 0.01 vs. sham group, †P < 0.05 ‚ ‡P < 0.01 vs. pacing group. LAV max, LA maximal volume; LAVmin, LA minimal volume; LAEF, LA ejection fraction; LAAVmax, LAA maximal volume; LAAVmin, LAA minimal volume; LAAEF, LAA ejection fraction. 5 weeks refers to 5 weeks post-operation

β3-AR stimulation exacerbated atrial myocyte apoptosis and myocardial interstitial fibrosis in the rapid pacing canine model of AF

Representative histological staining results are shown in Fig. 3. Compared to the sham group, the percentage of TUNEL-positive cells (apoptosis index, AI) (5.72 ± 4.41 vs. 21.15% ± 12.3%, P < 0.01) and the CVF (3.00± 1.18% vs. 10.29 ± 1.84%, P < 0.01) were markedly increased in the pacing group. β3-AR stimulation increased atrial myocyte apoptosis (29.71± 10.38% vs. 21.15 ± 12.31%, P < 0.01) and myocardial interstitial fibrosis (12.88 ± 3.77% vs. 10.29 ± 1.84%, P < 0.01) compared to the pacing group. In contrast, β3-AR inhibition reversed the damage to atrial myocytes (13.83± 4.61% vs. 21.15 ± 12.31%, P < 0.01) as well as the effects on interstitial fibrosis (8.01± 2.07% vs.10.29 ± 1.84%, P < 0.01).

Fig. 3

(A) Assay of apoptosis in the atrial myocardium. TUNEL-positive (apoptotic) nuclei are stained dark brown, while healthy nuclei are stained blue. The magnification is 200 x. (B) Apoptotic index (AI%) in the atrial myocardium of the sham, pacing, β3-AGO and β3-ANT groups. (C) Masson's trichrome stain in the atrial myocardium of the sham, pacing, β3-AGO and β3-ANT groups. Collagen fibers are stained blue, and cardiomyocytes are stained red. The magnification is 200 x. (D) Collagen volume fraction (CVF%) in atrial myocardium of the sham, pacing, β3-AGO and β3-ANT groups. Note the increased atrial AI and CVF in the pacing group compared to the sham group. An increase in the AI and CVF was observed in the β3-AGO group, whereas a decrease in the AI and CVF was observed in the β3-ANT group compared to the pacing group. **P < 0.01 vs. sham group; ##P < 0.01 vs. pacing group. β3-AGO refers to the β3-AR agonist BRL37344 group, and β3-ANT refers to the β3-AR antagonist L748337 group.

Fig. 3

(A) Assay of apoptosis in the atrial myocardium. TUNEL-positive (apoptotic) nuclei are stained dark brown, while healthy nuclei are stained blue. The magnification is 200 x. (B) Apoptotic index (AI%) in the atrial myocardium of the sham, pacing, β3-AGO and β3-ANT groups. (C) Masson's trichrome stain in the atrial myocardium of the sham, pacing, β3-AGO and β3-ANT groups. Collagen fibers are stained blue, and cardiomyocytes are stained red. The magnification is 200 x. (D) Collagen volume fraction (CVF%) in atrial myocardium of the sham, pacing, β3-AGO and β3-ANT groups. Note the increased atrial AI and CVF in the pacing group compared to the sham group. An increase in the AI and CVF was observed in the β3-AGO group, whereas a decrease in the AI and CVF was observed in the β3-ANT group compared to the pacing group. **P < 0.01 vs. sham group; ##P < 0.01 vs. pacing group. β3-AGO refers to the β3-AR agonist BRL37344 group, and β3-ANT refers to the β3-AR antagonist L748337 group.

Close modal

β3-AR stimulation deteriorated atrial myocardial ultrastructure in the rapid pacing canine model of AF

Representative electron micrographs highlighting the ultrastructural changes in the atria are shown in Fig. 4. Atrial myocytes in the sham group presented with regular sarcomere organization and nuclear morphology as well as abundant mitochondria with normal morphology and clear cristae (Fig. 4A). Following the four-week atrial pacing protocol, ultrastructural damage could be readily observed in the atria and was characterized by sparsely arranged myofilaments that appeared in disarray as well as mitochondria swelling with decreased cristae (Fig. 4B). Moreover, the β3-AR agonist BRL37344 worsened the chronic tachypacing-induced ultrastructural defects (Fig. 4C). In contrast, the β3-AR antagonist L748337 suppressed the ultrastructural damage (Fig. 4D).

Fig. 4

Electron micrographs of the atrial ultrastructure in the sham, pacing, β3-AGO and β3-ANT groups. (A) Sham group: Note the regular sarcomere organization, normal nuclear morphology, abundant mitochondria with clear cristae and distinct intercalated discs. (B) Pacing group: Note the disorganized and sparsely arranged myofilaments, mitochondria swelling with decreased cristae and increased interstitial connective tissue. (C) β3-AGO group: Note the severe disarray and dissected myofilaments with evident mitochondria swelling and vacuolation as well as increased interstitial connective tissue. (D) β3-ANT group: Note the significantly improved ultrastructural changes in atria with limited disorganized myofilaments, mitochondria swelling and fibrous tissue infiltration. The magnification is 8,000x (A‚C) and 10,000 x (B, D). Mit, mitochondria; Myo, myofilaments; Nuc, nucleus; Dis, intercalated discs. β3-AGO refers to the β3-AR agonist BRL37344 group, and β3-ANT refers to the β3-AR antagonist L748337 group.

Fig. 4

Electron micrographs of the atrial ultrastructure in the sham, pacing, β3-AGO and β3-ANT groups. (A) Sham group: Note the regular sarcomere organization, normal nuclear morphology, abundant mitochondria with clear cristae and distinct intercalated discs. (B) Pacing group: Note the disorganized and sparsely arranged myofilaments, mitochondria swelling with decreased cristae and increased interstitial connective tissue. (C) β3-AGO group: Note the severe disarray and dissected myofilaments with evident mitochondria swelling and vacuolation as well as increased interstitial connective tissue. (D) β3-ANT group: Note the significantly improved ultrastructural changes in atria with limited disorganized myofilaments, mitochondria swelling and fibrous tissue infiltration. The magnification is 8,000x (A‚C) and 10,000 x (B, D). Mit, mitochondria; Myo, myofilaments; Nuc, nucleus; Dis, intercalated discs. β3-AGO refers to the β3-AR agonist BRL37344 group, and β3-ANT refers to the β3-AR antagonist L748337 group.

Close modal

β3-AR stimulation increased superoxide anion production and reduced cardiac NO production in the rapid pacing canine model of AF

Compared to the sham group, the level of atrial superoxide anion increased in the rapid pacing canine model (1721.0 ± 483.5 a.u. vs. 1161.0 ± 370.3 a.u., P < 0.01) (Fig. 5A and B), suggesting a reaction to oxidative stress. Atrial NO production was decreased in the pacing group (204.4 ± 34.36 µmol/g protein vs. 279.2 ± 14.09 µmol/g protein, P < 0.01) compared to the sham group, as measured by the sum of the concentrations of the NO metabolites (nitrate and nitrite) (Fig. 5C). In contrast, the β3-AR agonist BRL37344 aggravated the atrial oxidative stress, as evidenced by the increase in the superoxide anion level (2470 ± 392.5 a.u. vs. 1721.0 ± 483.5 a.u., P < 0.01), as the NO generation was decreased (147.7± 16.61 µmol/g protein vs. 204.4 ± 34.36 µmol/g protein, P < 0.01) in the pacing canines compared to the pacing group with no drug. Again, the β3-AR antagonist L748337 suppressed these biochemical alterations (superoxide anion, 938.5 ± 232.4 a.u. vs. 1721.0 ± 483.5 a.u., P < 0.01; NO, 248.8 ± 20.20 µmol/g protein vs. 204.4 ± 34.36 µmol/g protein, P < 0.05).

Fig. 5

(A) Representative images of superoxide anion production in the left atrium as assayed based on DHE fluorescence in the sham, pacing, β3-AGO and β3-ANT groups. Red fluorescence indicates superoxide anion production. (B) Quantitative analysis of superoxide production in the sham, pacing, β3-AGO and β3-ANT groups. **P < 0.01 vs. Sham group, ##P < 0.01 vs. Pacing group. (C) Nitrate/nitrite represents NO production and was measured by the Griess assay in the sham, pacing, β3-AGO and β3-ANT groups. **P < 0.01 vs. sham group, #P <0.05, ##P < 0.01 vs. pacing group. (D) BH4 levels in the left atria of the sham, pacing, β3-AGO and β3-ANT groups. **P < 0.01 vs. sham group, ##P < 0.01 vs. pacing group. BH4: tetrahydrobiopterin. (E) nNOS, iNOS and eNOS mRNA expression levels in the sham, pacing, β3-AGO and β3-ANT groups. *P < 0.05 vs. sham group, ##P < 0.01, #P < 0.05 vs. pacing group. (F) Western blot analysis of nNOS, iNOS and eNOS expression using specific nNOS, iNOS and eNOS antibodies. (G) nNOS, iNOS and eNOS protein expression in the sham, pacing, β3-AGO and β3-ANT groups. *P < 0.05 vs. sham group, #P < 0.05 vs. pacing group. β3-AGO refers to the β3-AR agonist BRL37344 group, and β3-ANT refers to the β3-AR antagonist L748337 group.

Fig. 5

(A) Representative images of superoxide anion production in the left atrium as assayed based on DHE fluorescence in the sham, pacing, β3-AGO and β3-ANT groups. Red fluorescence indicates superoxide anion production. (B) Quantitative analysis of superoxide production in the sham, pacing, β3-AGO and β3-ANT groups. **P < 0.01 vs. Sham group, ##P < 0.01 vs. Pacing group. (C) Nitrate/nitrite represents NO production and was measured by the Griess assay in the sham, pacing, β3-AGO and β3-ANT groups. **P < 0.01 vs. sham group, #P <0.05, ##P < 0.01 vs. pacing group. (D) BH4 levels in the left atria of the sham, pacing, β3-AGO and β3-ANT groups. **P < 0.01 vs. sham group, ##P < 0.01 vs. pacing group. BH4: tetrahydrobiopterin. (E) nNOS, iNOS and eNOS mRNA expression levels in the sham, pacing, β3-AGO and β3-ANT groups. *P < 0.05 vs. sham group, ##P < 0.01, #P < 0.05 vs. pacing group. (F) Western blot analysis of nNOS, iNOS and eNOS expression using specific nNOS, iNOS and eNOS antibodies. (G) nNOS, iNOS and eNOS protein expression in the sham, pacing, β3-AGO and β3-ANT groups. *P < 0.05 vs. sham group, #P < 0.05 vs. pacing group. β3-AGO refers to the β3-AR agonist BRL37344 group, and β3-ANT refers to the β3-AR antagonist L748337 group.

Close modal

β3-AR stimulation decreased BH4 levels, increased iNOS expression and modulation of nNOS and eNOS in the rapid pacing canine model ofAF

We showed that atrial BH4 generation (0.218 ± 0.030 pmol/mg protein vs. 0.118 ± 0.021 pmol/mg protein, P < 0.01) (Fig. 5D) and GCH-1 expression (both mRNA and protein) (P < 0.05) (Fig. 6) were decreased in the pacing groups compared to the sham control group. Furthermore, β3-AR stimulation reduced atrial BH4 production (0.050 ± 0.007 pmol/mg protein vs. 0.118 ± 0.021 pmol/mg protein, P < 0.01) (Fig. 5D) and GCH-1 expression (P < 0.01) (Fig. 6) in the pacing β3-AGO group, whereas the β3-antagonist L748337 attenuated these effects (BH4, 0.173 ± 0.029 pmol/mg protein vs. 0.118 ± 0.021 pmol/mg protein, P < 0.01; Fig. 5D) (GCH-1 upregulation by L748337 is shown in Fig. 6; P < 0.01).

Fig. 6

GCH-1 mRNA and protein levels in atria of the sham, pacing, β3-AGO and β3-ANT groups. (A) GCH-1 mRNA levels in atria of the sham, pacing, β3-AGO and β3-ANT groups. *P < 0.05 vs. sham group, ##P < 0.01 vs. pacing group. (B) Western blot analysis of GCH-1 protein expression in the sham, pacing, β3-AGO and β3-ANT groups using specific GCH-1 antibodies (C) Quantitative analysis of GCH protein in all groups. **P < 0.01 vs. sham group, ##P < 0.01 vs. pacing group.

Fig. 6

GCH-1 mRNA and protein levels in atria of the sham, pacing, β3-AGO and β3-ANT groups. (A) GCH-1 mRNA levels in atria of the sham, pacing, β3-AGO and β3-ANT groups. *P < 0.05 vs. sham group, ##P < 0.01 vs. pacing group. (B) Western blot analysis of GCH-1 protein expression in the sham, pacing, β3-AGO and β3-ANT groups using specific GCH-1 antibodies (C) Quantitative analysis of GCH protein in all groups. **P < 0.01 vs. sham group, ##P < 0.01 vs. pacing group.

Close modal

Both iNOS mRNA and protein expression were increased in the pacing group compared to the sham group (P < 0.05) (Fig. 5E, F and G). iNOS expression was also increased by the β3-AR agonist and suppressed by the β3-AR antagonist in the pacing groups compared to pacing alone (P < 0.05).

We further demonstrated that nNOS expression (mRNA and protein) was increased (P < 0.05) and eNOS (mRNA and protein) expression was decreased (P < 0.05) in the pacing groups compared to the sham group (Fig. 5E, F, and G). Parallel to other findings, the β3-AR agonist BRL37344 markedly reduced eNOS expression (P < 0.05), whereas the β3-AR antagonist L748337 reversed these effects (P < 0.05) (Fig. 5E, F, and G). Interestingly, nNOS levels did not vary among the pacing groups in the presence of the β3-AR agonist and antagonist (P > 0.05) (Fig. 5E, F, and G).

β3-AR stimulation resulted in superoxide anion generation, which could be decreased by the specific iNOS inhibitor

Using a superoxide colorimetric assay, we demonstrated that atrial homogenates had increased superoxide production in the β3-AR agonist group (0.064 ± 0.008 µmol/mg protein vs. 0.099 ± 0.024 µmol/mg protein, P < 0.01) and that these effects were inhibited in the β3- AR antagonist group (0.046 ± 0.006 µmol/mg protein vs. 0.064 ± 0.008 µmol/mg protein, P < 0.01) (Fig. 7), which was consistent with the DHE fluorescence results (Fig. 5B). Interestingly, pretreatment of the atrial homogenates with 100 µM of the specific iNOS inhibitor 1400W abolished the increase in superoxide production observed with β3-AR agonist stimulation (0.062 ± 0.025 µmol/mg protein vs. 0.099 ± 0.024 µmol/mg protein, P < 0.01) (Fig. 7).

Fig. 7

Quantitative analysis of superoxide anion production in the sham, pacing, β3-AGO and β3-ANT groups using a colorimetric assay. (A) Comparisons of superoxide levels were directly assayed after tissue collection in the sham, pacing, β3-AGO and β3-ANT groups and after incubation with the iNOS inhibitor 1400w. **P < 0.01. (B) Comparisons of total superoxide levels amongst all groups. The results were subtracted from the total levels to obtain the iNOS-dependent superoxide levels. **P < 0.01 vs. sham group, ##P < 0.01 vs. pacing group.

Fig. 7

Quantitative analysis of superoxide anion production in the sham, pacing, β3-AGO and β3-ANT groups using a colorimetric assay. (A) Comparisons of superoxide levels were directly assayed after tissue collection in the sham, pacing, β3-AGO and β3-ANT groups and after incubation with the iNOS inhibitor 1400w. **P < 0.01. (B) Comparisons of total superoxide levels amongst all groups. The results were subtracted from the total levels to obtain the iNOS-dependent superoxide levels. **P < 0.01 vs. sham group, ##P < 0.01 vs. pacing group.

Close modal

This present study revealed for the first time that β3-AR activation exacerbates atrial structural remodeling by activating iNOS uncoupling/oxidative stress, which could be considered a novel therapeutic target for AF

β3-AR activation promoted AF by aggravating atrial structural remodeling and dysfunction

β3-AR belongs to a group of G protein-coupled receptors characterized by seven transmembrane domains having features that determine their resistance to long-term downregulation and significance in regulating cardiac performance following acute and chronic stress. β3-AR levels were upregulated, which aggravated cardiac dysfunction during heart failure [10,11]. Additionally, in humans and dogs, β3-AR is detected in lung and peripheral arteries and causes vasodilation [22,23]. Therefore, β3-AR had beneficial effects in perinephritic hypertension [24]. Meanwhile, β3-AR stimulation has antiarrhythmic effect in canine ventricular tachycardia [25]. Different levels of β3-AR in the cardiac chambers at different stages of the pathologies and various downstream pathways may determine the functional characterization of β3-AR in different cardiovascular diseases.

In our study, we observed an upregulation of β3-AR in AF canines induced by rapid atrial pacing. β3-AR stimulation further increased atrial apoptosis and interstitial fibrosis, indicative of an exacerbation of atrial structural remodeling. Atrial structural remodeling in turn promotes AF development and self-persistence. Apoptosis and interstitial fibrosis during atrial structural remodeling are both considered potential therapeutic targets for AF [26,27,28,29,30,31]. Subsequent apoptosis and reparative fibrosis replaces dead cardiomyocytes, which interrupts fiber bundle continuity, thereby slowing conduction [29,32,33]. Therefore, atrial structural remodeling has a strong impact on electrical remodeling. Our study also indicated that β3-AR activation increased AF inducibility and prolonged AF duration, which might be associated with the exacerbating effect of β3-AR on atrial structural remodeling. Yu and colleagues recently highlighted that β3-AR activation promotes atrial electrical remodeling by decreasing the abundance of L-type calcium channels as well as increasing the inward rectifier potassium current (IK1) and transient outward potassium current (Ito) in acute AF models of rabbit and atrial myocytes [12]. Furthermore, Liu et al. recently demonstrated that β3-AR activation disturbed cardiac energy metabolism and aggravated AF in a rabbit AF model [34]. Moreover, in this present study, therapies using the β3-AR antagonist L748337 attenuated atrial apoptosis and interstitial fibrosis in AF canines, which prevented AF development.

Generally, atrial dilatation enhances cardiac vulnerability to AF, which in turn results in progressive atrial dilatation. Therefore, AF and atrial dilatation are mutually promoting and constitute a vicious circle. Atrial dilatation results in contractile dysfunction associated with typical features of AF [35]. Atrial size is a key determinant of AF reentry [36]. Therefore, therapies targeted at reducing atrial size may prevent AF. Treatment with the β3-AR antagonist L748337 in our AF model resulted in decreased atrial volume and attenuation of impaired atrial function, which was effective in preventing AF.

β3-AR activation increased iNOS uncoupling and oxidative stress

Oxidative stress is a major underlying pathology that promotes AF [13]. Excess ROS can result in a loss of enzyme function, mitochondrial dysfunction and cellular death, all of which strongly correlate with atrial structural remodeling [13]. Thus, inhibition of cardiac ROS may be effective in circumventing AF. Of the numerous cellular sources of ROS, uncoupled NOS is the major source in the human heart [13,37].

NOS uncoupling is involved in many pathologies, such as diabetes mellitus, hypertension, ageing and atherosclerosis [38], all of which were associated with AF. In the process of NO production, the L-arginine concentration does not affect the reaction [39,40,41], although low BH4 bioavailability relative to NOS results in uncoupled NOS and excessive superoxide production [14]. In this present study, we observed a decrease in BH4 levels, an increase in atrial myocardial superoxide anion levels and a decrease in NO production by β3-AR stimulation in the AF canine model. This indicates that β3-AR activation induces NOS uncoupling and oxidative stress.

We measured endogenous changes in the three isoforms of NOS following β3-AR stimulation in AF canines to investigate which isoform dominated the effects of β3-AR on NOS uncoupling. The heart normally expresses the constitutive enzymes nNOS and eNOS, whereas iNOS is inducibly expressed in pathological states [15,42]. Additionally, iNOS is closely associated with inflammation [15] and oxidative stress [37], both of which are involved in the atrial structural remodeling in AF [43]. Han and colleagues also reported that iNOS protein levels are increased in the atria of patients with permanent AF [44].

β3-AR mediates negative inotropic effects on the ventricular myocardium by eNOS activation [45,46]. In contrast, β3-AR activation increases the contractility of the human right atrium [45]. Moreover, we revealed that BRL37344 inhibited eNOS expression in the atrium of AF canines. Different effects of β3-AR on the atria and ventricle occur potentially due to lower β3-AR expression in the atria [47] and different activation mechanisms of β3-AR on eNOS [48]. Additionally, β3-AR activation mediated anti-hypertrophic and antioxidant effects on hypertrophic ventricular cardiomyocytes via a nNOS-dependent mechanism [49]. In contrast, our data revealed that BRL3 7344 activated iNOS but did not influence nNOS levels in the AF canine model. Moreover, the specific iNOS inhibitor 1400W abolished the induction of superoxide production by the agonist of β3-AR, which validated our previous hypothesis. Consequently, mediation of different signaling pathways may determine the diverse performance of β3-AR in atria and ventricles. Therefore, β3-AR stimulation aggravated atrial oxidative stress, which likely resulted from iNOS activation. The importance of iNOS was further validated by our β3-AR antagonist, L748337, which reversed the aberrant expression of iNOS isoforms.

BH4 is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase, with GCH-1 acting as the rate-limiting enzyme [14]. Therefore, BH4 concentrations strongly correlate with GCH-1 expression. This present study showed that BH4 down-regulation by BRL37344 may occur due to reduced levels of GCH-1. Animal studies have confirmed that BH4 supplementation can reduce oxidative stress [50] and attenuate cardiac remodeling post-infarction [51]. However, due to the high cost and poor feasibility of BH4 in clinical practice, it is not utilized here in this study. β3-AR inhibition with L748337 in this study increased GCH-1 and BH4 levels, indicating a more feasible therapy for reducing atrial oxidative stress and structural remodeling in AF.

In conclusion, our study demonstrates that β3-AR activation triggers a cascade of events resulting in atrial structural remodeling (Fig. 8), and these effects might be partially attributed to iNOS uncoupling and oxidative stress in AF.

Fig. 8

Schemata of the mechanisms underlying the role of β3-AR stimulation in atrial structural remodeling associated with AF.

Fig. 8

Schemata of the mechanisms underlying the role of β3-AR stimulation in atrial structural remodeling associated with AF.

Close modal

The β3-AR antagonist L748337 was clearly effective in suppressing the progression of atrial structural remodeling, and the specific iNOS inhibitor attenuated atrial oxidative stress. Thus, both mediators have an important effect on the arrhythmogenic substrate for AF and could have therapeutic roles in clinical practice. Therefore, this present study revealed two targets for AF treatment.

AF (Atrial fibrillation); AI (Apoptotic index); B (Biopterin); BH4 (Tetrahydrobiopterin); BH2 (Dihydrobiopterin); DHE (Cell-permeable dihydroethidium); β3-AR (β3 adrenergic receptor); β3-AGO (β3 adrenergic receptor agonist); β3-ANT (β3 adrenergic receptor antagonist); CVF (Collagen volume fraction); eNOS (Endothelial NOS); GCH-1 (Guanosine triphosphate cyclohydrolase-1); HPLC (High-performance liquid chromatography); HE (Hydroethidium); HRP (Horseradish peroxidase); iNOS (Inducible NOS); LA (Left atrium); LAA (Left atrial appendage); LAVmax (LA maximal volume); LAAVmax (LAA maximal volume); LAVmin (LA minimal volume); LAAVmin (LAA minimal volume); LAEF (LA ejection fraction); LAAEF (LAA ejection fraction); NO (Nitric oxide); nNOS (Neuronal NOS); NOS (Nitric oxide synthase); NADPH (Nicotinamide adenine dinucleotide phosphate); ROS (Reactive oxygen species); TBST (Tris-buffered saline and tween 20); WB (Western blot); PVDF (Polyvinylidenedifluoride); O2- (Superoxide anion).

This work was supported by the National Natural Science Foundation of China (grant numbers: 81270252, 30971251, 81070160 and 81100071), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81121003), the Scientific Research Fund of Heilongjiang Provincial Education Department (grant number 12521206) and grants from the Science and Research Foundation of the First Hospital of Harbin.

1.
Camm AJ, Lip GY, De Caterina R, Savelieva I, Atar D, Hohnloser SH, Hindricks G, Kirchhof P: 2012 focused update of the ESC Guidelines for the management of atrial fibrillation: an update of the 2010 ESC Guidelines for the management of atrial fibrillation. Developed with the special contribution of the European Heart Rhythm Association. Eur Heart J 2012;33:2719-2747.
2.
Krogh-Madsen T, Abbott G, Christini D: Effects of electrical and structural remodeling on atrial fibrillation maintenance: a simulation study. PLoS Comput Biol 2012;8:e1002390.
3.
Liu L, Geng J, Zhao H, Yun F, Wang X, Yan S, Ding X, Li W, Wang D, Li J, Pan Z, Gong Y, Tan X, Li Y: Valsartan reduced atrial fibrillation susceptibility by inhibiting atrial parasympathetic remodeling through MAPKs/Neurturin pathway. Cell Physiol Biochem 2015;36:2039-2050.
4.
Liu Y, Geng J, Yang LSB, Liu Y, Li Y, Cheng C, Shen J, Li W: β3-adrenoceptor mediates metabolicprotein remodeling in a rabbit model oftachypacing-induced atrial fibrillation. Cell Physiol Biochem 2013;32:1631-1642.
5.
Iwasaki Y-k, Nishida K, Kato T, Nattel S: Atrial fibrillation pathophysiology: Implications for management. Circulation 2011;124:2264-2272.
6.
Tan AY, Zimetbaum P: Atrial fibrillation and atrial fibrosis. J Cardiovasc Pharmacol 2011;57:625-629.
7.
Dong J, Zhao J, Zhang M, Liu G, Wang X, Liu Y, Yang N, Liu Y, Zhao G, Sun J, Tian J, Cheng C, Wei L, Li Y, Li W: β3-adrenoceptor impairs mitochondrial biogenesis and energy metabolism during rapid atrial pacing-induced atrial fibrillation. J Cardiovasc Pharmacol Ther 2016;21:114-126.
8.
Gauthier C, Rozec B, Manoury B, Balligand J-L: Beta-3 adrenoceptors as new therapeutic targets for cardiovascular pathologies. Curr Heart Fail Rep 2011;8:184-192.
9.
Gauthier C, Sèze-Goismier C, Rozec B: Beta 3-adrenoceptors in the cardiovascular system. Clin Hemorheol Microcirculat 2007;37:193-204.
10.
Moniotte S, Kobzik L, Feron O, Trochu JN, Gauthier C, Balligand JL: Upregulation of beta(3)-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium. Circulation 2001;103:1649-1655.
11.
Cheng HJ, Zhang ZS, Onishi K, Ukai T, Sane DC, Cheng CP: Upregulation of functional beta(3)-adrenergic receptor in the failing canine myocardium. Circ Res 2001;89:599-606.
12.
Yu J, Li W, Li Y, Zhao J, Wang L, Dong D, Pan Z, Yang B: Activation of beta(3)-adrenoceptor promotes rapid pacing-induced atrial electrical remodeling in rabbits. Cell Physiol Biochem 2011;28:87-96.
13.
Sovari AA, Dudley Jr. SC: Reactive oxygen species-targeted therapeutic interventions for atrial fibrillation. Front Physiol 2012;3:311.
14.
Alkaitis MS, Crabtree MJ: Recouplingthe cardiac nitric oxide synthases: tetrahydrobiopterin synthesis and recycling. Curr Heart Fail Rep 2012;9:200-210.
15.
Forstermann U, Sessa WC: Nitric oxide synthases: regulation and function. Eur Heart J 2012;33:829-837, 837a-837d.
16.
Li Y, Li W, Gong Y, Li B, Liu W, Han W, Dong D, Sheng L, Xue J, Zhang L, Chu S, Yang B: The effects of cilizapril and valsartan on the mRNA and protein expressions of atrial calpains and atrial structural remodeling in atrial fibrillation dogs. Basic Res Cardiol 2007;102:245-256.
17.
Swartz MF, Fink GW, Lutz CJ, Taffet SM, Berenfeld O, Vikstrom KL, Kasprowicz K, Bhatta L, Puskas F, Kalifa I, Jalife J: Left versus right atrial difference in dominant frequency, K(+) channel transcripts, and fibrosis in patients developing atrial fibrillation after cardiac surgery. Heart Rhythm 2009;6:1415-1422.
18.
Sanders P, Berenfeld O, Hocini M, Jais P, Vaidyanathan R, Hsu LF, Garrigue S, Takahashi Y, Rotter M, Sacher F, Scavee C, Ploutz-Snyder R, Jalife J, Haissaguerre M: Spectral analysis identifies sites of high-frequency activity maintaining atrial fibrillation in humans. Circulation 2005;112:789-797.
19.
Zhao J, Li J, Li W, Li Y, Shan H, Gong Y, Yang B: Effects of spironolactone on atrial structural remodelling in a canine model of atrial fibrillation produced by prolonged atrial pacing. Br J Pharmacol 2010; 159:1584-1594.
20.
Khan M, Mohan IK, Kutala VK, Kumbala D, Kuppusamy P: Cardioprotection by sulfaphenazole, a cytochrome p450 inhibitor: mitigation of ischemia-reperfusion injury by scavenging of reactive oxygen species. J Pharmacol Exp Ther 2007;323:813-821.
21.
Fukushima T, Nixon JC: Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 1980;102:176-188.
22.
Dessy C, Moniotte S, Ghisdal P, Havaux X, Noirhomme P, Balligand JL: Endothelial beta3-adrenoceptors mediate vasorelaxation of human coronary microarteries through nitric oxide and endothelium-dependent hyperpolarization. Circulation 2004;110:948-954.
23.
Tagaya E, Tamaoki J, Takemura H, Isono K, Nagai A: Atypical adrenoceptor-mediated relaxation of canine pulmonary artery through a cyclic adenosine monophosphate-dependent pathway. Lung 1999;177:321-332.
24.
Donckier JE, Massart PE, Mechelen HV, Heyndrickx GR, Gauthier C, Balligand JL: Cardiovascular effects of beta 3-adrenoceptor stimulation in perinephritic hypertension. Eur J Clin Invest 2001;31:681-689.
25.
Zhou S, Tan AY, Paz O, Ogawa M, Chou C-C, Hayashi H, Nihei M, Fishbein MC, Chen LS, Lin S-F, Chen P-S: Antiarrhythmic Effects of Beta3-adrenergic receptor stimulation in a canine model of ventricular tachycardia. Heart Rhythm 2008;5:289-297.
26.
Wakili R, Voigt N, Kaab S, Dobrev D, Nattel S: Recent advances in the molecular pathophysiology of atrial fibrillation. J Clin Invest 2011;121:2955-2968.
27.
Burstein B, Qi XY, Yeh YH, Calderone A, Nattel S: Atrial cardiomyocyte tachycardia alters cardiac fibroblast function: a novel consideration in atrial remodeling. Cardiovasc Res 2007;76:442-452.
28.
Nattel S, Harada M: Atrial remodeling and atrial fibrillation: recent advances and translational perspectives. J Am Coll Cardiol 2014;63:2335-2345.
29.
Iwasaki YK, Nishida K, Kato T, Nattel S: Atrial fibrillation pathophysiology: implications for management. Circulation 2011;124:2264-2274.
30.
Nattel S, Burstein B, Dobrev D: Atrial remodeling and atrial fibrillation: mechanisms and implications. Circ Arrhythm Electrophysiol 2008;1:62-73.
31.
Yue L, Xie J, Nattel S: Molecular determinants of cardiac fibroblast electrical function and therapeutic implications for atrial fibrillation. Cardiovasc Res 2011;89:744-753.
32.
Burstein B, Nattel S: Atrial fibrosis: mechanisms and clinical relevance in atrial fibrillation. J Am Coll Cardiol 2008;51:802-809.
33.
Burstein B, Comtois P, Michael G, Nishida K, Villeneuve L, Yeh YH, Nattel S: Changes in connexin expression and the atrial fibrillation substrate in congestive heart failure. Circ Res 2009;105:1213-1222.
34.
Liu Y, Geng J, Liu Y, Li Y, Shen J, Xiao X, Sheng L, Yang B, Cheng C, Li W: beta3-adrenoceptor mediates metabolic protein remodeling in a rabbit model of tachypacing-induced atrial fibrillation. Cell Physiol Biochem 2013;32:1631-1642.
35.
Schotten U, Neuberger HR, Allessie MA: The role of atrial dilatation in the domestication of atrial fibrillation. Prog Biophys Mol Biol 2003;82:151-162.
36.
Zou R, Kneller J, Leon LJ, Nattel S: Substrate size as a determinant of fibrillatory activity maintenance in a mathematical model of canine atrium. Am J Physiol Heart Circ Physiol 2005;289:H1002-1012.
37.
Rochette L, Lorin J, Zeller M, Guilland JC, Lorgis L, Cottin Y, Vergely C: Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: possible therapeutic targets? Pharmacol Ther 2013;140:239-257.
38.
Alkaitis MS, Crabtree MJ: Recouplingthe cardiac nitric oxide synthases:Tetrahydrobiopterin synthesis and recycling. Curr Heart Fail Rep 2012;9:200-210.
39.
Pollock J, Forstermann U, Mitchell J: Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and nativebovine aortic endothelial cells. Proc Natl Acad Sci 1991;88:10480-10484.
40.
Closs E, Scheld J, Sharafi M: Substrate supply for nitric-oxide synthase in macrophages and endothelial cells: role of cationic amino acid transporters. MolPharmacol 2000;57:68-74.
41.
Simon A, Plies L, Habermeier A: Role of neutral amino acid transport and proteinbreakdown for substrate supply of nitric oxide synthase in human endothelial cells. CircRes 2003;93:813-820.
42.
Heusch P, Aker S, Boengler K, Deindl E, van de Sand A, Klein K, Rassaf T, Konietzka I, Sewell A, Menazza S, Canton M, Heusch G, Di Lisa F, Schulz R: Increased inducible nitric oxide synthase and arginase II expression in heart failure: no net nitrite/nitrate production and protein S-nitrosylation. Am J Physiol Heart Circ Physiol 2010;299:H446-453.
43.
Li J, Solus J, Chen Q, Rho YH, Milne G, Stein CM, Darbar D: Role of inflammation and oxidative stress in atrial fibrillation. Heart Rhythm 2010;7:438-444.
44.
Han W, Fu S, Wei N, Xie B, Li W, Yang S, Li Y, Liang Z, Huo H: Nitric oxide overproduction derived from inducible nitric oxide synthase increases cardiomyocyte apoptosis in human atrial fibrillation. Int J Cardiol 2008;130:165-173.
45.
Skeberdis VA, Gendviliene V, Zablockaite D, Treinys R, Macianskiene R, Bogdelis A, Jurevicius J, Fischmeister R: beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current. J Clin Invest 2008;118:3219-3227.
46.
Gauthier C, Seze-Goismier C, Rozec B: Beta 3-adrenoceptors in the cardiovascular system. Clin Hemorheol Microcirc 2007;37:193-204.
47.
Pott C, Brixius K, Bundkirchen A, Bolck B, Bloch W, Steinritz D, Mehlhorn U, Schwinger RH: The preferential beta3-adrenoceptor agonist BRL 37344 increases force via beta1-/beta2-adrenoceptors and induces endothelial nitric oxide synthase via beta3-adrenoceptors in human atrial myocardium. Br J Pharmacol 2003;138:521-529.
48.
Brixius K, Bloch W, Pott C, Napp A, Krahwinkel A, Ziskoven C, Koriller M, Mehlhorn U, Hescheler J, Fleischmann B, Schwinger RH: Mechanisms of beta 3-adrenoceptor-induced eNOS activation in right atrial and left ventricular human myocardium. Br J Pharmacol 2004;143:1014-1022.
49.
Watts V, Sepulveda F, Cingolani O, Ho A, Niu X, Kim R, Miller K, Vandegaer K, Bedja D, Gabrielson K, Rameau G, O'Rourke B, Kass D, Barouch L: Anti-hypertrophic and anti-oxidant effect of beta3-adrenergic stimulation in myocytes requires differential neuronal nos phosphorylation. J Mol Cell Cardiol 2013;62:8-17.
50.
Moens AL, Claeys MJ, Wuyts FL, Goovaerts I, Van Hertbruggen E, Wendelen LC, Van Hoof VO, Vrints CJ: Effect of folic acid on endothelial function following acute myocardial infarction. Am J Cardiol 2007;99:476-481.
51.
Masano T, Kawashima S, Toh R, Satomi-Kobayashi S, Shinohara M, Takaya T, Sasaki N, Takeda M, Tawa H, Yamashita T, Yokoyama M, Hirata K: Beneficial effects of exogenous tetrahydrobiopterin on left ventricular remodeling after myocardial infarction in rats: the possible role of oxidative stress caused by uncoupled endothelial nitric oxide synthase. Circ J 2008;72:1512-1519.
Open Access License / Drug Dosage / Disclaimer
This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.